Monitoring and Manipulating Cellular Transmembrane Potentials using Nanostructures

ABSTRACT

The use of nanostructures to monitor or modulate changes in cellular membrane potentials is disclosed. Nanoparticles having phospholipid coatings were found to display improved responses relative to nanoparticles having other coatings that do not promote localization or attraction to membranes.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to Provisional PatentApplication Ser. No. 60/659,975 filed Mar. 8, 2005, the contents ofwhich are incorporated herein by reference,

FIELD OF THE INVENTION

The invention relates to compositions and methods useful for monitoringand manipulating cellular transmembrane voltages. In particular,nanoparticles and their use in monitoring and manipulating transmembranevoltages is disclosed.

DESCRIPTION OF RELATED ART

All cells have phospholipid membranes that serve as bimolecular barriersand to separate cell contents from the extracellular environment. Thepurpose of the plasma membrane is to maintain the necessary differencein composition between two compartments by restricting or permitting thepassage of materials through the membrane as a function of intracellularsignaling,

Each cell has a resting membrane potential originating from theso-called “separation of charge” across the normally impermeablephospholipid hilayer. Because of unequal distribution of positively andnegatively charged ions in the extracellular and intracellularcompartments, all living cells have a negative resting membranepotential, ranging from −5 mV to 100 mV. The ion permeability of theplasma membrane is determined by the presence of ion channels,transmembrane proteins specialized in passive ion transport. Membranepotential can be changed by changing membrane permeability to as certainion in response to an activating stimulus, thus allowing a flux of ionsdown their electrochemical gradient. Transport of ions across themembrane through ion channels will lead to disturbance of the existingequilibrium of ion concentrations on both sides of membrane and, thus,to changes of electrical properties of the cell.

Cells communicate with each other through changes in membrane potential,Therefore, monitoring the cellular membrane potential and its changesallows monitoring of cell viability and function.

Ion channels are transmembrane proteins present in both excitable andnon-excitable cells. Ion channels permit and regulate movement andconduction of ions down their electrochemical gradients across anormally ion-impermeant lipid bilayer. They produce electrical signalsleading to action potential generation that controls a number of keyprocesses, including neuronal signaling, heart beat, brain function,sensory transduction and muscle contraction. In addition to setting theresting membrane potential and controlling cellular excitability, thesetransmembrane proteins play important roles affecting the physiologicalstate of cells by being involved in cell proliferation, hormonesecretion and homeostasis of water and electrolytes.

Activation of ion channels by any mechanism results in redistribution(changes in concentrations) of intracellular extracellular ions andconsequent change in cellular membrane potential. Thus, recordingchanges in membrane potential allows direct monitoring of ion channelactivity,

A typical organism has hundreds to thousands of different types of ionchannels, while an individual cell can have ten to twenty differenttypes. Each ion channel exhibits high selectivity for one or a few ionspecies.

Different ion channel families are classified based on their activationstimuli, selectivity to different ions, inactivation mechanisms andpharmacological profiles. There are voltage-activated andligand-activated ion channels. Superfamilies of voltage-gated sodium,potassium, calcium, and chloride ion channels have been defined usingelectrophysiological, pharmacological and molecular techniques; they arenamed according to their selective permeability for a particular cationwith reference to their voltage dependence, kinetic behavior ormolecular identity. Superfamilies of ligand-gated channels are much lessstructurally related and named after their activation ligand, i.e.cyclic nucleotide-gated channels or GABA channels.

Functionally, the opening or closing of ion channels can be controlledor “gated” by the binding of signaling molecules (ligand-gatedchannels), by a change in the membrane potential (voltage-gatedchannels), or by mechanical stimulation (mechanosensitive channels) thatresults in conformational changes within ion channel structures leadingto opening of a pore and allowing a flux of ions inside or outside thecell.

For example, a voltage-gated sodium channel is closed at a restingmembrane potential below −60 mV, and opens upon depolarization of themembrane (i.e., a shift in the membrane potential to a less negativevalue).

The structures of voltage-gated sodium, calcium and potassium channelshave common functional elements. All ion channels are transmembraneproteins comprised of several homologous repeats arranged around acommon ion-selective aqueous pore that opens in response to anactivating stimulus that allows ions to enter or exit the cell. Eachrepeat consists of six transmembrane domains (S1-S6) with the S4 domainplaying the specialized role of voltage sensor. Channel opening andclosing (“gating”) is controlled by this voltage sensitive domain of theprotein containing charged amino acids that respond to changes in theelectric field. Translocation of a voltage sensitive domain leads toconformational changes in the structure of the channel resulting inconducting (open/activated) or non-conducting (closed/inactivated)states.

Although ligand-gated channels differ significantly from one another,there are two structural elements present in every channel: aligand-binding domain and a pore domain. Binding of a specific ligandtriggers conformational changes leading to opening of the pore domainand allowing the ion flux into/out of the cell, which is reflected in achange in transmembrane potential.

The various states of ion channel activation provide uniqueopportunities for more efficient drug discovery, enablingstate-dependent molecules to be developed that, for example, only bindto non-conducting (inactivated) channels. A desirable goal is to targetdrugs to tissues exhibiting abnormal electrical activity, while leavingnormal channels in active tissues unaffected. Also, identifying new ionchannels, testing their functions, and validating them as drug targetsare current efforts of many biotech companies and academic researchers.

Ion Channels in Drug Discovery

Ion channels are of particular importance in the pharmaceutical industryin two areas: ion channels as drug targets and ion channel safetypharmacology. Ion channels are significant targets in the drug discoveryprocess, generating several billion dollars in sales per annum. Abnormalion channel function or ion channel expressions have been linked to anumber of therapeutic areas (i.e. cardiac arrhythmia, hypertension,epilepsy, pain). Ion channel modulator drugs for these have yet to bedeveloped. Many pharmaceutical companies have active ion channel drugdevelopment projects or programs. Additionally, a number of biotech orbiopharmaceutical companies focus exclusively on ion channel drugdevelopment (ChanTest, Cleveland, Ohio; BioFocus, Cambridge, UK; Icagen,Durham, N.C.).

Ion channels are involved in many vital functions, and any dysfunctionof ion channels caused by changes in biochemical regulation, expressionlevels, or structural mutations can impact the well-being of livingorganisms. In humans, inherited or induced changes in ion channelfunction could result in serious complications to health. Severaldisease states are related to dysfunctional ion channels. Ion channeldefects produce a clinically diverse set of disorders that vary fromcystic fibrosis and some finis of migraine to renal tubular defects andepisodic ataxias.

In particular, ion channels have been implicated in cardiac arrhythmias,familial periodic paralyses, cystic fibrosis, epilepsy, diabetes,asthma, angina pectoris, malignant hypertherrnia, pain, hypertension,epilepsy, etc. Ion channels represent key molecular targets for drugdiscovery. Pharmaceutical and biotechnology companies have successfullytargeted ion channels in their bid to make new more effective drugs. Nowvarious ion channel blockers or openers are being used and evaluated astherapeutic drugs for a variety of diseases.

Voltage-gated calcium ion channels are involved in numerous cellularfunctions, and their role in generating a defined disease phenotype iscomplex. Certain types of calcium-channels may play a role innociception and migraine pathophysiology. In human medicine,calcium-channel blockers are being evaluated for, among other things,treating glaucoma, deep vein thrombosis, and pulmonary hypertension, inrenal transplantation, and for prevention of perfusion injury.

Several voltage-dependent calcium channels blockers have been shown tobe effective in inhibiting pain, Furthermore, blockage of so-callednon-L-type calcium channels was found to exert therapeutic effects inthe treatment of severe pain and ischemic stroke.

Dysfunction of potassium channels has been associated with thepathophysiology of a number of neurological, as well as peripheral,disorders (e.g., episodic ataxia, epilepsy, neurmyotonia, Parkinson'sdisease, congenital deafness, long QT syndrome).

Activation of potassium ion channels generally reduces cellularexcitability, making potassium-channel openers potential drug candidatesfor the treatment of diseases related to hyperexcitability such asepilepsy, neuropathic pain, and neurodegeneration.

Most notably, mutations of the HERO potassium ion channels expressed incardiac tissues or pharmacological blockage of HERO channels cause heartdisease (long Q-T syndrome), which leads to increased risk ofventricular tachycardia and sudden death. Several drugs affect thesechannels and can lead to life threatening cardiac arrhythmias. In thisperspective, drug discovery companies usually find it necessary toevaluate each of their drug candidates for interference with thesechannels. Thus, many companies conduct HERO testing before any furtherinvestigation is carried out.

The dynamic nature of sodium ion channel expression makes them importanttargets for pharmacological manipulation in the search for new therapiesfor pain. For example, mutations in the gene encoding the alpha subunitof sodium-channels have been linked to paroxysmal disorders such asepilepsy, long QT syndrome, hyperkalemic periodic paralysis in humansand to motor endplate disease and cerebellar ataxia in mice.Voltage-gated sodium ion channel have been shown to be key mediators ofthe pathophysiology of pain. One of the most frequently used anestheticdrugs used is Lidocaine which inhibits sodium ion channels. Changes inbrain sodium-channels may be a cause of central pain, and further,abnormal expression of sodium-channel genes and its contributions tohyperexcitability of primary sensory neurons have been discussed.Recently, sensory-neuron-specific (SNS) TTX-resistant sodium-channelshave been examined for their role in nociception and pain. This studysuggests that blockage of SNS expression or function may produceanalgesia.

Experimental Approaches for Ion Channel Research

The preferred method for studying ion channels is the patch clamp method(Neher, E. and Sakmann, B., Nature 260(5554): 799-802 (1976); Hamill, OP., et al., Plugers Arch. 391(2): 85-100(1981)).

This technique consists of contacting a cell with the tip of a veryclean glass micropipette (diameter of about 1 μm), and obtaining a highresistance seal (leakage resistance >1 GOhm, GigaSeal) between the glassand the cell surface by applying gentle suction. Next, by applyinggreater suction or a large voltage, it is possible to break theintra-pipette portion of membrane and thereby make direct electricalcontact between the cell interior and the pipette electrode (whole-cellconfiguration of patch clamp method). Different voltages can then beapplied to the pipette electrode, and the currents measured representthe current through the cell membrane, which includes the integralcurrent through the ion channels present.

To date, the patch-clamp method has been considered the industry goldstandard for monitoring ion channel activity. The patch-clamp directlyrecords ion channel activity, has sub-millisecond temporal resolution,very high information content and is extremely sensitive including theability to study “single” ion channels. Due to its high informationcontent, patch-clamp-based screening has very low rate of “falsenegatives” and “false positives”.

Although this technique allows detailed biophysical characterization ofion channel activation, inactivation, gating, ion selectivity, and druginteractions, throughput is quite low and ease-of-use of patch-clampinstrumentation is generally unsatisfactory for effective massscreening. The demands of ion channel high throughput screening (“HTS”)include robust instrumentation and high signal-background ratio combinedwith satisfactory ease-of-use. Historically, ion channel HTS is equatedwith low information content, emphasizing the need for novel rapid andeasy methods in which more useful information can he gathered aboutmembrane potential changes in various cell types.

Ion Channel HTS Approaches

Traditionally, HTS technologies employed for ion channel primaryscreening rely on binding assays, ion-flux assays and fluorometrieapproaches. Until now the most significant task for these methods hasbeen to generate enough HTS data with acceptable information content andreliability.

The search for molecules that modulate ion channel function has beenhindered by the lack of direct electrical measurements in HTS formats,Membrane excitability in cell-based assays is a dynamic phenomenon thatrequires fast, precise and accurate measurements to gather highinformation content data. Real-time measurements of transmembranepotential kinetics that accurately reflect ion channel activity arefundamental to cell physiology, but are difficult to measure in existingHTS format methods.

Reliable and robust high HTS assays for ion channels are important inion-channel based drug discovery. Ion channels are dynamic proteins, andtherefore require assays that “sense” their various functional states.Competition-binding assays, although successfully used for other targetclasses, often fail to identify ligands that modulate specific ionchannel states. Cell-based functional assays, therefore, are preferredfor FITS of ion channel targets.

Currently Available Assays

Modem technologies employed for ion channel screening include: bindingassays, ion flux assays, fluorometric imaging and electrophysiology.

Binding assays for cell surface receptors are used in screen developmentand primary screening. This type of assay is frequently carried outusing scintillation proximity assay (SPA) or fluorescence detectiontechniques, which have replaced the older radiolabelled ligands andfiltration assays. The SPA technique relies upon excitation of ascintillant microbead upon binding of a radiolabelled ligand to areceptor immobilized on the surface of the bead.

Fluorescence spectrometry is used to measure the binding equilibriumbetween a fluorophore-labeled ligand and receptor. Unfortunately bindingassays only detect binding of compounds to ion channels and do notreveal changes in target function, such as modulation of ion channelkinetics.

Optical readouts of ion channel function are favorable for HTS becausethey are versatile, amenable to miniaturization and automation andpotentially sensitive.

Fluorescence readouts are used widely both to monitor intracellular ionconcentrations and to measure membrane potentials. For example, largetransient increases in intracellular calcium concentration throughactivation of ion channels can be monitored using fluorescent probessuch as Fluo-3 and Calcium Green. In addition to ion-selectivefluorescent indicators, there are several fluorescent dyes that aresensitive to changes in membrane potential, including styryl, bisoxonol,and fluorescence resonance energy transfer--based voltage-sensitivedyes.

For example, the fluorescent dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol, or DiBAC4(3), has been the, reagent of choice formeasuring membrane potential in HTS formats. Redistribution of the dyein the cellular membrane as a result of depolarizing or hyperpolarizingstimuli in cells causes changes in fluorescence. However, utilization ofDiBAC4(3) has several limitations, including slow kinetics (in theseconds to minutes range) and fluctuations in response to changes intemperature and concentration of the dye. In addition, screeningexperiments using bisoxonol dyes require multi--step procedures and take30-60 min for dye loading, potentially compromising the fidelity andreducing throughput of DiBAC4(3)-based screening assays.

HTS Patch-Clamp

The patch clamp technique is widely used to study currents through ionchannels. The whole-cell patch-clamp is used today in tertiary screeningof selected lead molecules in late stages of the drug discovery process.Whole-cell patch-clamp, however, is not suitable for initial highthroughput screening.

Although very powerful, this technique is labor-intensive and,therefore, limited to few data point measurements per day. This lowthroughput has encouraged the use of other less specific and lesssensitive technologies for high-throughput screening of ion channeltargets.

Optimal HTS Ion Channel Assay Requirements

In high-throughput screening campaigns (200,000+ samples), bindingassays remain the first choice in terms of throughput and cost. Thisreflects the technical ease of performing these types of assays and,hence, their ability to he automated. However, in modern ion channeldrug discovery screening, there is a trend toward use of cellular-basedfunctional assays as primary screening tools.

Cellular functional assays are used as primary or secondary assays todetermine functionality of compounds from a binding screen and also toassess toxicity. These types of assays are information rich andtherefore potentially of significant value in drug discovery.

Identifying targets and putative drug candidates by obtaining as muchknowledge as possible per experiment about the effects of each compoundis the ultimate goal for initial ion channel screening.

Voltage Sensitive Probes

Currently used fluorescent voltage sensor dyes, which respond topotential-dependent accumulation and redistribution across the cellularmembrane, are limited to steady-state assays of membrane potential. Thisis because the fluorescence response of these dyes occurs minutes afterthe change in membrane potential. Since voltage sensor dyes are chargedthey also interfere with the membrane potential caused by the ioniccurrent; to reduce this signal-to--noise effect the dye concentrationhas to be kept below a certain level. Thus, redistribution-based voltagesensor dyes are prone to false-negatives. In addition, compound-voltagedye interactions can show high false-positive rates.

Voltage-sensing Fluorescence Resonance Energy Transfer (FRET) acceptors,for example coumarin-tagged phospholipids integrated into the cellmembrane ameliorate many of the problems associated with standardvoltage sensors, allowing sub-second kinetic determination, Using highthroughput screening FRET-based voltage sensors a throughput of several96-well plates per hour can be performed with the Voltage Ion ProbeReader (VIPR™), a product developed by Aurora BioSciences (now VertexPharmaceuticals, Inc.; Cambridge, Mass.).

Compared with results obtained with traditional patch-clamp method, VIPRassays are less sensitive. The temporal resolution in fluorescence-basedion-channel assays using voltage-sensor dyes reduces the accessiblekinetic range relative to patch-clamp-based ion channel assays.

Ion-specific fluorescent probes for intracellular ions have been shownto be useful for ion channel screening. Depending on the application, anumber of different dyes are available with different ranges ofaffinities, of which fluorescent calcium indicators are the mostcommonly used. A significant disadvantage of calcium dye-basedion-channel assays is their slow kinetic resolution of changes ofintracellular calcium concentration, due to uncontrolled orunpredictable cellular processes. This can interfere with assay results.To achieve high throughput and low noise, FLIPR-type fluorescent readersare commonly used in conjunction with calcium specific dyes. So far onlyassays involving measurements of calcium channel activity or othernon-selective cation channels have proven to be robust enough foreffective FITS efforts.

In summary, an optimal FITS ion channel screening method would have hightemporal resolution, high sensitivity and high information content,resulting in low rates of “false negatives” and “false positives”.Despite the materials and methods available to study ion channels, thereexists a need for new materials and methods that are easy, robust, anduseful.

Nanoparticles or Nanocrystals

Numerous studies have been published describing nanoparticles andmethods for their use. Semiconductor nanocrystals are sometimes referredto as “Quantum Dots” or “QDots”, although these are registeredtrademarks of Quantum Dot Corporation (a wholly owned subsidiary ofInvitrogen Corp.; Carlsbad, Calif.). Nanopartieles are typicallyspherical or nearly so, having a central core, a surrounding shell, andoptional capping groups, linkers, and other surface-conjugatedmaterials.

Semiconductor nanoparticles are nanometer-scale crystals composed ofhundreds to thousands of atoms of an inorganic semiconductor material inwhich electron-hole pairs can he created and confined.

Specific optical properties of nanoparticles are based on the mechanismof quantum confinement. Quantum confinement is the trapping of electronsor electron “holes” (charge carriers) in a space small enough that theirquantum (wave-like) behavior dominates their classical (particle-like)behavior.

In nanoparticles, where motions of electrons holes are highly limited inthree dimensional space, quantum confinement results in a strongincrease of optical excitation energies compared to the bulksemiconductor material. For quantum confinement to occur, the dimensionof the confining device or particle must he comparable to theelectron-hole Bohr radius of the material it is made from. Afterelectron-hole pairs in the core of a nanocrystal are formed uponexcitation with light, they can recombine and re-emit light having anarrow and symmetric emission spectrum that depends directly on the sizeof the crystal. The smaller the nanoparticle core, the bigger thebandgap between the valence and conduction bands, the bluer the emittedphoton; and vice versa (redder emission) for larger nanoparticles.

Commercially available semiconductor nanocrystals are comprised ofseveral layers, including a core, an inorganic lattice-matchingcrystalline shell (to improve the nanocrystal's optical properties andpossibly serving to minimize cytotoxicity), and a coating or coatings(to allow water compatibility and for effective interaction withmodifiers such as biomacromolecules).

Nanocrystals are used in information technology (the quantum computer),light emitting diodes, lasers, and telecommunication devices, barcoding, photodetectors, optical switching, and thermoelectric devices.Recently, nanocrystals have been used for cell labeling, cell tracking,in vivo imaging, DNA detection, protein labeling, and in other detectionmodes.

Nanocrystals have excellent optical properties as biological opticalsensors, including size-tunable emission, narrow spectral width, broadexcitation spectrum, high quantum yields, high two-photon cross-section,and low photobleaching rates.

However, until now biological and biotechnological applications ofnanocrystals have been mostly limited to their use as biomarkers ratherthan as detectors of biological processes.

Applications of nanocrystals in industry include, for example,nanocrystal-based electro-luminescent devices capable of emitting lightof various wavelengths in response to external stimuli, where variationsin applied voltage could result in change of color of the light emittedby the device.

Many patents and patent publications report nanocrystal compositions,methods for their preparation, and methods for their use. The followingcollection is a sampling of the research done to date.

U.S. Pat. No. 5,505,928 (issued Apr. 9, 1996) describes methods ofpreparing III-V semiconductor nanocrystal materials. Examples of suchmaterials include GaAs, GaP, GaAs-P, GaSb, InAs, InP, InSb, AlAs, AlP,and AlSb. The produced materials can be 1-6 nm in size, and arerelatively monodisperse.

U.S. Pat. No. 5,990,479 (issued Nov. 23, 1999) describes nanocrystalslinked to affinity molecules. Listed affinity molecules includemonoclonal and polyclonal antibodies, nucleic acids, proteins,polysaccharides, and small molecules such as sugars, peptides, drugs,and ligands.

U.S. Pat. No. 6,114,038 (issued Sep. 5, 2000) describes water soluble,functionalized nanocrystals having a capping compound of the formulaHS(CH₂)_(n)X, wherein X is a carboxylate. The nanocrystals also have adiaminocarboxylic acid which is operably linked to the capping compound.

U.S. Pat. No. 6,207,229 (issued Mar. 27, 2001) describes a coatednanocrystal capable of light emission includes a substantiallymonodisperse rianoparticle selected from the group consisting of CdX,where X═S, Se, or Te; and an overcoating of ZnY, where Y═S, or Sc.Methods of preparing the nanocrystals using a first semiconductor coreand a precursor capable of ther conversion into a second semiconductormaterial that forms a coating layer over the core.

U.S. Pat. No. 6,207,392 (issued Mar. 27, 2001) describes semiconductornanocrystals having one or more attached /inking agents. Thenanocrystals can include nanocrystals of Group II-VI semiconductors suchas MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe,ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, and HgTe as well as mixedcompositions thereof; as well as nanocrystals of Group III-Vsemiconductors such as GaAs, inGaAs, InP, and InAs.

U.S. Pat. No. 6,251,303 (issued Jun. 26, 2001), U.S. Pat. No. 6,319,426(issued Nov. 20, 2001) and U.S. Pat. No. 6,444,143 (issued Sep. 3, 2002)describe a water-soluble semiconductor nanocrystal. The outer layer ofthe nanocrystal contains a molecule having at least one linking groupfor attachment of the molecule to the overcoating shell layer, and atleast one hydrophilic group optionally spaced apart from the linkinggroup by a hydrophobic region sufficient to prevent electron chargetransfer across the hydrophobic region.

U.S. Pat. No. 6,274,323 (issued Aug. 14, 2001) describes a method ofdetecting a polynucleotide in a sample, using a semiconductornanocrystal in an immunosorbent assay.

U.S. Pat. No. 6,306,610 (issued Oct. 23, 2001) describes semiconductornanocrystals having attached multidentate ligands. The nanocrystals canbe associated with various biological molecules such as proteins andnucleic acids.

U.S. Pat. No. 6,322,901 (issued Nov. 27, 2001) describes monodispersecoated nanocrystals that emit light in a spectral range of no greaterthan about 60 nm full width at half max (FWHM). The spectral range ofthe nanocrystals is about 470 nm to about 620 nm, and the particle sizeof the nanocrystal core is about 20 angstroms to about 125 angstroms.

U.S. Pat. No. 6,326,144 (issued Dec. 4, 2001) describes semiconductornanocrystals linked to various compounds using a linker of structureH_(g)X((CH₂)_(n)CO₂H)_(y) and salts thereof, where X is S, N, P or O═P;n is greater than or equal to 6; and z and y are selected to satisfy thevalence requirements of X.

U.S. Pat. No. 6,423,551 (issued Jul. 23, 2002) and U.S. Pat. No.6,699,723 (issued Mar. 2, 2004) describe a water soluble semiconductornanocrystal having a linking agent capable of linking to an affinitymolecule. A list of affinity molecules includes monoclonal andpolyclonal antibodies, nucleic acids (both monomeric and oligomeric),proteins, polysaccharides, and small molecules such as sugars, peptides,drugs, and ligands. Examples of linking agents includeN-(3-aminopropyl)3-mercapto-benzamide, 3-aminopropykrimethoxysilane,3-mercaptopropyl-trimethoxysilane, 3-maleirnidopropyl-trimethoxysilane,and 3-hydrazidopropyl-trimethoxysilane,

U.S. Pat. No. 6,426,513 (issued Jul. 30, 2002) describes a water-solublesemiconductor nanocrystal comprising a quantum dot having a selectedband gap energy; an overcoating layer comprising a material having aband gap energy greater than the band gap energy of the quantum dot; andan outer layer comprising a compound having a formula, SH(CH₂)_(n)X,where X is carboxylate or sulfonate, and n is greater than or equal to8.

U.S. Pat. No. 6,500,622 (issued Dec. 31, 2002) describes semiconductornanocrystals having attached polynucleotide sequences. The nanocrystalscan be used to determine the presence or absence of a target sequence ina sample. The nanocrystal can be identified using a spectral code.

U.S. Pat. No. 6,548,168 (issued Apr. 15, 2003) describes a method ofstabilizing particles with an insulating, semiconducting and/or metalliccoating. A particle-coating admixture containing a bifunctional ligandis used to bind a particle to the coating. Examples of bifunctionalligands include 3-mercaptopropyl trimethoxysilane (“MPS”),1,3-propanedithiol, 3-aminopropanethiol (“APT”), and3-aminopropyltrimethoxysilamine (“APS”).

U.S. Pat. No. 6,576,291 (issued Jun. 10, 2003) describes a method ofmanufacturing a nanocrystallite, the method comprising contacting ametal, M, or an M-containing salt, and a reducing agent to form anM-containing precursor, M being Cd, Zn, Mg, Hg, Al, Ga, In, or TI;contacting the M-containing precursor with an X donor, X being O, S, Se,Te, N, P, As, or Sb to foi in a mixture; and heating the mixture in thepresence of an amine to form the nanocrystallite. The nanocrystallitescan be used in a variety of applications including optoelectronicdevices including electroluminescent devices such as light emittingdiodes (LEDs) or alternating current thin film electroluminescentdevices (ACTFELDs).

U.S. Pat. No. 6,649,138 (issued Nov. 18, 2003) describes awater-dispersible nanoparticle comprising: an inner core comprised of asemiconductive or metallic material; a water-insoluble organic coatingsurrounding the inner core; and, surrounding the water-insoluble organiccoating, an outer layer comprised of a multiply amphipathic dispersantmolecule, wherein the dispersant molecule comprises at least twohydrophobic regions and at least two hydrophilic regions. Thenanoparticles can be conjugated to various affinity molecules, allowinguse in applications such as fluorescence immunocytochemistry,fluorescence microscopy, DNA sequence analysis, fluorescence in situhybridization (FISH), fluorescence resonance energy transfer (FRET),flow cytometry (Fluorescence Activated Cell Sorter; FACS) and diagnosticassays for biological systems.

U.S. Pat. No. 6,815,064 (issued Nov. 9, 2004) describes a nanoparticlecontaining a Group 2 element, a Group 12 element, a Group 13 element, aGroup 14 element, a Group 15 element, a Group 16 element, Fe, Nb, Cr,Mn, Co, Cu, or Ni in an inorganic, shell around the semiconductor core.The compositions and methods of preparation are proposed to facilitatethe overgrowth of a high-quality, thick shell on a semiconductive coreby compensating for the mismatching of lattice structures between thecore and shell materials.

Despite the materials and methods available to study ion channels, thereexists a need for new materials and methods that are easy, robust, anduseful. Additionally, there is a need for methods of controlling themembrane potential of cells to facilitate studying the effects ofadministered materials.

SUMMARY OF THE INVENTION

The use of nanostructures to measure or modulate changes in cellular orsubcellular membrane potentials is disclosed. Nanostructures associatedwith cells respond to changes in membrane potential, and can be easilymonitored. The methods can be used to monitor the effects of addedexternal agents on cellular membrane potential.

DESCRIPTION OF THE FIGURES

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these figures in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows the effect of electrophysiological stimulation on cellscontaining intracellular nanoparticles. The upper trace shows the changein fluorescence intensity of semiconductor nanocrystals inside the cellin response to change in transmembrane potential using the patch-clampmethod, The x-axis is time in seconds; the y-axis is Arbitrary Units,Semiconductor nanocrystals were loaded inside cell through patchpipette. The lower trace represents the corresponding voltagestimulation protocol along the same time scale.

FIG. 2 shows the effect of electrophysiological stimulation on cellscontaining intracellular lipid-modified nanoparticles. The upper tracesshow the change in fluorescence intensity of phospholipid-functionalizedsemiconductor nanocrystals in response to change in transmembranepotential using the patch-clamp method from three areas of interest (2cells and background). Cell #1 was exposed to voltage stimulation, whilecell #2 was not exposed to voltage stimulation. Semiconductornanocrystals were applied to the extracellular side of the cellularmembrane. The lower trace represents the corresponding voltagestimulation protocol along the same time scale.

FIG. 3 shows the results of monitoring transient changes in cells causedby addition of a high concentration of potassium chloride (100 mM). Thecircle, diamond, triangle, and square symbols represent differentregions of interest (ROI).

DETAILED DESCRIPTION OF THE INVENTION

While compositions and methods are described in teams of “comprising”various components or steps (interpreted as meaning “including, but notlimited to”), the compositions and methods can also “consist essentiallyof” or “consist of” the various components and steps, such terminologyshould be interpreted as defining essentially closed-member groups.

Methods of Assaying Changes in Transmembrane Potential

One embodiment of the invention is directed towards methods for assayinga change in transmembrane potential. The methods can comprise providingat least one target, wherein the target is a cell, cellular fraction, orartificial membrane structure; contacting the target with at least onenanostructure to form a treated target; stimulating the treated target;assaying emission from the nanostructure; and correlating the emissionwith the change in transmembrane potential. An optional additional stepcan comprise assaying emission from the nanostructure after thecontacting step but before the stimulating step. This additional stepcan act as a “control” or “blank” measurement.

The target can be one or more intact cells, can be one or more cellularfractions, or one or more artificial membrane structures. Examples ofcellular fractions include any luminal organelles such as nucleus,ribosomes, mitochondria, endoplasmic reticulum, Golgi apparatus,vacuoles, synaptic vesicles and lysosomes. Examples of the artificialmembrane structures include phospholipid micelles, micro- andnanocapsules and semi-liquid films supportive structures. The contactingstep can comprise introducing the nanostructure into the target.Alternatively, the contacting step can comprise introducing thenanostructure into a cellular membrane of the target. The nanostructurecan alternatively be introduced onto or near a cellular membrane of thetarget. Nanostructures “near” the target are sufficiently close inproximity so as to be able to detect changes in transmembrane potential.As an example, nanostructures closer than about 100 microns aresufficiently near a target so as to have this property.

The target can be stimulated by a wide variety of methods. Examples ofsuch stimulation methods include electrical stimulation, magneticstimulation, chemical stimulation, biological stimulation, orcombinations thereof Examples of electrical stimulation include the useof a patch clamp, and application of an external electric field.Examples of chemical stimulation include contacting the target with apotassium salt or a sodium salt, or with diMrent types of intramembranepore-forming molecules. Examples of biological stimulation includeactivating the target with a light-sensitive ion channel, or contactingthe target with the chemical entities, acting as modifiers of ionchannel activity. Examples of magnetic stimulation include activatingthe target with alternating electromagnetic field of the appropriatefrequency and amplitude.

Targets can be electrically stimulated by a variety of methods. Onestimulation protocol (voltage amplitudes and duration of stimulation) isoften chosen based on activation kinetics of the ion channel ofinterest, For example, targets can be maintained at a first membranepotential voltage, subjected to a depolarizing pulse at a secondmembrane potential voltage, and returned to the first membrane potentialvoltage. The second membrane potential voltage is typically morepositive than the first membrane potential voltage, but it is possiblethat the first membrane potential voltage is more positive than thesecond membrane potential voltage. For example, the first membranepotential voltage can be negative, while the second membrane potentialvoltage can be positive. An example is −70 mV for the first membranepotential voltage, and +40 mV for the second membrane potential voltage.Alternatively, the first or second membrane potential voltage can be 0mV. Examples include −200 mV for the first membrane potential voltage,and 0 mV for the second membrane potential voltage. An additionalexample is 0 mV for the first membrane potential voltage, and 200 mV forthe second membrane potential voltage. Specific examples of firstmembrane potential voltages and second membrane potential voltages canbe independently selected from about −200 mV, about −180 mV, about −160mV, about −140 mV, about −120 mV, about −100 mV, about −80 mV, about −60mV, about −40 mV, about −20 mV, about 0 mV, about 20 mV, about 40 mV,about 60 mV, about 80 mV, about 100 mV, about 120 mV, about 140 mV,about 160 mV, about 180 mV, about 200 mV, and ranges between any two ofthese values.

Alternatively, more complicated voltage patterns can be used in themethods. The methods can further comprise exposing the targets to atleast one step voltage prior to subjecting them to the depolarizingpulse at a second membrane potential voltage. The step voltage is anintermediate voltage between the first membrane potential voltage andthe second membrane potential voltage. The step voltage can be used tomeasure leak subtraction. For example, a first membrane potentialvoltage of −80 mV, a step voltage of −50 mV, and a second membranepotential voltage of 20 mV can be used.

The depolarizing pulse can generally be applied for any length of time.For example, the depolarizing pulse can be applied for up to about 5000seconds. Examples of the length of time include about 10 microseconds,about 1 milliseconds, about 10 milliseconds, about 100 milliseconds,about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about5 seconds, about 10 seconds, about 20 seconds, about 30 seconds, about40 seconds, about 50 seconds, about 60 seconds, about 70 seconds, about80 seconds, about 90 seconds, about 100 seconds, about 500 seconds,about 1000 seconds, about 2000 seconds, about 3000 seconds, about 4000seconds, about 5000 seconds, and ranges between any two of these values.

The one or more cells can generally be any type of cells which have amembrane and membrane potential. For example, the cells can be bacterial(Gram-positive or Gram-negative), eucaryotic, procatyotic, fungal,insect, avian, reptilian, oocyte, fly, zebrafish, nematode, fish,amphibian, or mammalian cells. The methods can also be used on non-cellmaterials such as artificial membranes, liposomes, and phospholipidbilayers. Examples of primary mammalian cells include human, mouse, rat,dog, cat, bear, moose, cow, horse, pig, or Chinese hamster ovary (“CHO”)cells. Other examples of types of cells include immune system cells(e.g., B-cells, T-cells), oocytes, red blood cells, white blood cells,neurons, epithelial, glia, fibroblast, cancer cells, and immortalizedcells.

The nanostructures can be introduced into the target by a number ofmethods. Examples of such methods include use of a patch pipette,passive or active uptake via endocytosis or other uptake mechanisms,electroporation, liposome-mediated delivery, pluronic blockcopolymer-mediated delivery, cell-penetrating peptide-mediated uptake,protein-mediated uptake, microinjection, transfection, viral delivery,optoporation, pore-forming substrates, membrane intercalators, orcombinations thereof.

Methods of nanostructures loading into the cellular membrane (or otherkinds of membranes mentioned above) include the immobilization of thenanostructures onto the supportive structures (for example, onto thebottom of a well in the microtiter plate) and subsequent addition ofsolution containing cells to an experimental chamber (such as amicrotiter plate well).

The nanostructures can generally be any nanostructures. Examples ofnanostructures include a nanocrystal, a film, a nanowire, a patternedsubstrate, and a mesh. Nanoparticles can generally be any nanoparticles.Semiconductor nanoparticles or nanocrystals typically have asemiconductor core, a sha, and optionally, one or more surfacetreatments. Semiconductor nanoparticles are commercially available fromcompanies such as Quantum Dot Corp. (a wholly owned subsidiary ofInvitrogen Corp.; Carlsbad, Calif.) and Evident Technologies (Troy,N.Y.). There also exist many published descriptions of the preparationof nanoparticles.

The semiconductor core and shell can independently be made of a materialof an element from Group 2 or 12 of the Periodic Table of the Elements,and an element selected from Group 16 of the Periodic Table of theElements. Examples of such materials include ZnS, ZnSe, ZnTe, CDs, CdSe,CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, Case, CaTe, SrS, SrSe, Sae,BaS, BaSe, and BaTe. Alternatively, the semiconductor core and shell canindependently be made of a material made of an element from Group 13 ofthe Periodic Table of the Elements, and an element from. Group 15 of thePeriodic Table of the Elements. Examples of such materials include GaN,GaP, GaAs, GaSb, InP, InAs, and InSb. Alternatively, the semiconductorcore and shell can independently be made of a material made of anelement from Group 14 of the. Periodic Table of the Elements. Examplesof such a material include Ge, and Si. Alternatively, the semiconductorcore and shell can independently be made of lead materials such as PbSor PbSe. The semiconductor core and shell can be made of alloys ormixtures of any of the above listed materials as well.

The semiconductor nanocrystal can generally be of any size (averagediameter), but typically are about 0.1 nm to 1000 nm in size. Morenarrow ranges of sizes include about 0.1 nm to about 1 nm, about 1 nm toabout 50 nm, and about 1 nm to about 20 nm. Specific size examplesinclude about 0.1 nm, about 0.5 nm, about 1 nm, about 2 nm, about 3 nm,about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm,about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, about 20 nm,about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about50 nm, and ranges between any two of these values.

A typical single-color preparation of nanoparticles has crystals thatare preferably of substantially identical size and shape. Nanocrystalsare typically thought of as being spherical or nearly spherical inshape, but can actually be any shape. Alternatively, the nanocrystalscan be non-spherical in shape. For example, the nanocrystal's shape canchange towards oblate spheroids for redder colors. It is preferred thatat least about 60%, at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, and ideally about 100% of the particlesare of the same size. Size deviation can be measured as root mean squareof the diameter, with less than about 10% root mean square beingpreferred. Size deviation can be less than about 10% ms, less than about9% ms, less than about 8% ms, less than about 7% ms, less than about 6%ms, less than about 5% ms, or ranges between any two of these values.Such a collection of particles is sometimes referred to as being“monodisperse”.

It is well known that the color (emitted light) of the semiconductornanocrystal can be “tuned” by varying the size and composition of thenanocrystal. Nanocrystals preferably absorb a wide spectrum ofwavelengths, and emit a narrow wavelength of light. The excitation andemission wavelengths are typically different, and non-overlapping. Thewidth of emission is preferably less than about 50 nm, and morepreferably less than about 20 nm at full width at half maximum of theemission band (FWHM). Examples of emission widths (FWHM) include about50 nm, about 40 nm, about 30 nm, about 20 nm, and about 10 nm. Theemitted light preferably has a symmetrical emission of wavelengths. Theemission maxima can generally be at any wavelength from about 200 nm toabout 2000 nm. Examples of emission maxima include about 200 nm, about400 am, about 600 nm, about 800 nm, about 1000 nm, about 1200 nm, about1400 nm, about 1600 nm, about 1800 nm, about 2000 nm, and ranges betweenany two of these values.

Nanoparticles can also have a metal core, and in some cases, asurrounding shell structure. The metal core. can be made from noblemetals. Examples of such metals include silver, gold, and copper.

The nanoparticles can have surface coatings adding variousfunctionalities. For example, the nanocrystals can be coated withlipids, phospholipids, fatty acids, polynucleic acids,polyethyleneglycol, primary antibodies, secondary antibodies, antibodyfragments, protein or nucleic acid based aptainers, biotin,streptavidin, proteins, peptides, small organic molecules, organic orinorganic dyes, precious or noble metal clusters.

Alternatively, the nanoparticles can be made from a range of inorganicmaterials, including silicon, alumina, zirconia, ceria, yttria andoxides of tin and zinc. For example, silicon nanoparticles possess manyof the advantageous features of compound semiconductor nanocrystals,such as size-tunable luminescence across the visible spectrum. Inaddition, silicon nanoparticles also low toxicity, highbiocornpatibility, efficient and stable surface functionalization, andpotential low cost.

The use of nanoparticles in ion channel assays has multiple desirablefeatures. Since nanoparticles have rapid response times, distinctivevoltage dependencies are difficult to unintentionally inactivate, andthe nanoparticles can provide a direct optical readout of voltagegradient changes across a membrane, The nanoparticles also possess otherdesirable qualities such as low toxicity, high photo-stability, theability to be used in multiplexing applications, and their ability to betargeted using conjugated or otherwise associated materials.

Spectral characteristics of nanoparticles can generally be monitoredusing any suitable light-measuring or light-accumulatinginstrumentation. Examples of such instrumentation are CCD(charge-coupled device) cameras, video devices, CIT imaging, digitalcameras mounted on a fluorescent microscope, photomultiplies,fluorometers and luminometers, microscopes of various configurations,and even the human eye. The emission can be monitored continuously or atone or more discrete time points. The photostability and sensitivity ofnanoparticles allow recording of changes in electrical potential overextended periods of time,

Additional methods of assaying the emission from the nanostructureinclude measuring changes in light intensity, light polarization, lightabsorption, color of the emission, emission lifetime or half-life, orthe “blinking” pattern,

An additional embodiment of the invention is directed towardsnanoparticles coated with phospholipids. An example of such ananocrystal is a commercially available phospholipid-coated MapleRed-Orange EviTag-T2 nanocrystal (Evident Technologies; Troy, N.Y.).There also exist published descriptions on preparation of lipid coatedsemiconductor nanocrystal materials.

Methods for the Excitation of Cells

An additional embodiment of the invention is directed towards the use ofrianostructures to control the transmembrane potential of cells. Opticalmethods are attractive for use in biological applications due to theirnon-invasive nature and ease of use. For example, photo-inducedelectrical excitation of neuronal cells has been demonstrated using afilm of semiconductor material (Frohmherz, P. and Stett, A., Phys. Rev.Lett. 75(8): 1670-1673 (1995); Starovoytov, A. et al., J. Veurophysiol.93(2): 1090-1098 (2005)). Neuronal cells were attached to a thin film ofa semiconductor material, achieving close contact of the extracellularmembrane and the semiconductor surface. Illumination of the substratewith a laser beam has been shown to electrically excite the cellsattached to the semiconductor surface.

Nanostructures such as nanoparticles exposed to light can act as agenerator of a local electromagnetic field in their vicinity. The effectis believed to be due to creation of free charge carriers (electron-holepairs upon illumination of nanoparticles) and consecutive chargeseparation. The currently proposed mechanism of action is electrostaticcoupling of the cellular membrane and the surface of semiconductor,effectively forming a capacitor. When nanoparticles are placed in closeproximity to a cell, the cumulative electromagnetic field generated byphoto-excited nanoparticles will interact with the cellulartransmembrane electrical gradient, resulting in an electromagnetic fieldthat dictates the cellular membrane potential. Local depolarization ofpart of cellular membrane may be sufficient to generate depolarizationin the whole cell.

In addition to use of the above described nanocrystals modifiednanoparticles can be used to achieve a strong, stable, and controllablelocal electric field. Such modifications include high surface charge(e.g. CdTe/CdSe as core/shell combination), doping nanoparticles withmaterials that would act as donors or acceptors of one type of freecharge carriers, creating nanoparticles with p- or n-type surface traps,conjugation of molecules that would contribute to a charge separation,and so on. Active generation of a cellular transmembrane potential canbe achieved through use of nanoparticles that can convert light intoelectric power.

In conventional solar cells, electron-hole pairs are created by lightabsorption in a semiconductor core, with charge separation andcollection accomplished under the influence of electric fields withinthe core.

As nanoparticles are approximately the same thickness as a cellularmembrane, insertion into the membrane exposes the poles of thenanoparticle to both the extra- and intracellular space. Uponillumination with light, nanoparticles become a path for free chargecarrier flow through the membrane, passing an electric current and inturn affecting the transmembrane potential. This way, voltage controlover the cell could be achieved by changing, for example, the incidentlight's intensity and/or polarization.

Nanoparticies can be synthesized in shapes of different complexity suchas spheres, rods, discs, triangles, nanorings, nanoshells, tetrapods,and so on. Each of these geometries have distinctive properties: spatialdistribution of the surface charge, orientation dependence ofpolarization of the incident light wave, and spatial extent of theelectric field. Non-uniform coating of nanoparticles with a dielectricmaterial (such as phospholipids) can also help guide the free chargecarriers from one side of the membrane to the other.

In order to manipulate free charge carrier concentration and mobility,nanoparticles can be doped with impurities such as indium, phosphorus,boron, and aluminum, and so on. A blend of nanoparticles and organicpolymers may be advantageous for this application as nanoparticles arehighly efficient in conducting electrons, whereas polymers are better atconducting holes. Functionalization of semiconductor nanoparticies withchromophores could also optimize this application by separating photonabsorption from free charge carrier transport.

Accordingly, methods for the optical control of the transmembranepotential of a target can comprise providing at least one target,wherein the target is a cell or cellular fraction; contacting the targetwith at least one nanostructure under conditions suitable forinteraction or insertion of the nanostructure with a cellular orsubcellular membrane to prepare a treated target; delivering energy tothe treated target; and detecting response of the target.

The cells can be any of the cells described above. The nanostructure canbe any nanostructure including any of the nanostructures describedabove.

The conditions suitable for interaction or insertion can include avariety of methods. Examples of such methods include passive or activeuptake via endocytosis, electroporation, liposome-inediated delivery,pluronic block copolymer-mediated delivery, cellpenetratingpeptide-mediated uptake, protein-mediated uptake, microinjection,transfection, viral delivery, optoporation, pore-forming substrates,membrane intercalators, or combinations thereof.

The delivering energy can include delivering light, electrical energy,magnetic energy, and so on. The delivering energy step can be performedby essentially any illumination method, including laser illumination,mercury lamp illumination, xenon lamp illumination, halogen lampillumination, LED illumination, and so on. An illuminating step ispreferably performed at a wavelength or wavelength range suitable forabsorption by the nanostructure.

The detecting step can be performed using a variety of methods using anysuitable light-measuring or light-accumulating instrumentation. Examplesof such instrumentation are a camera, a digital camera, a video camera,a CMOS camera, a CCD camera, a digital camera mounted on a fluorescentmicroscope, a photomultiplier, a fluorometer, a luminometer, amicroscope, and even the human eye. The cellular response can bemonitored continuously or at one or more discrete time points.

Alternatively, the detecting step can include use of a secondarydetection mechanism. An example of such a secondary detection mechanismis the use of fluorescence resonance energy transfer (“FRET”). WithFRET, the nanostructure can transfer its energy to a second moleculethat then emits a detectable signal. Additional secondary detectionmechanisms rely on changes in a cell that can be independently detected.For example, the cell may undergo lysis. Alternatively, the cell mayundergo a chemical change, increasing or decreasing the concentration ofone or more chemical or biochemical agents that can be independentlymeasured.

At least one additional material can be added to the at least one cellor to the treated cell to assay the cellular response to the additionalmaterial. For example, the cell can be first contacted with the at leastone nanoparticle, illuminated, and the cellular response detected as a“control” sample. The treated cell can then be contacted with theadditional material to prepare a material-treated cell, illuminated, anddetected. This second cellular response can be compared with the first(control) cellular response. .A difference between the first cellularresponse and the second cellular response would indicate whether theaddition of the material had any effect on cellula(behavior. A differentadditional material, or an additional dose of the same additionalmaterial can be added, followed by illumination and detection of a thirdcellular response. This can be done in a serial manner any number oftimes. For example, increasing dosages of a material can be detected,resulting in a third cellular response, a fourth cellular response, afifth cellular response, a sixth cellular response, and so on. Theseserial cellular responses can be plotted or otherwise compared, and theeffects of the serial treatments can be determined.

Alternatively, “control” and “test” samples can be performed inparallel. For example, a first cell can be contacted with ananoparticle, illuminated, and the control cellular response detected.In parallel, either serially or simultaneously, a second cell can becontacted with a nanoparticle and a test material, illuminated, and thetest cellular response detected. The control cellular response and thetest cellular response can be compared.

The at least one additional material can generally be any material.Examples of such materials include drug candidates, modulators ofcellular function, molecular moieties for enhanced drug delivery,molecular probes candidates, and so on.

Assay Materials

An additional embodiment of the invention is directed towards one ormore containers having a layer of nanostructures deposited on one ormore surfaces. For example, the container can be a test tube, centrithgetube, or microtiter plate (e,g., 96 or 384 well plate). The entire innersurface of the tube or plate's wells can be coated with thenanostructures mentioned above, Alternatively, the lower or bottom innersurface of the tube or wells can be coated with the nanostructures,These assay materials can be stored for subsequent use with cells orother biological or artificial membrane materials,

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor(s) to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the scope of theinvention.

EXAMPLES Example 1 Preparation of Cells on Coverslips

Experiments were perfoimed on A431 (a human cell line from an epidermoidcarcinoma) cells or CHO (Chinese hamster ovary) cells stably expressingM1 muscarinic G_(q)-protein coupled receptor using nanoparticlescommercially available from Quantum Dot Corp. (a wholly owned subsidiaryof Invitrogen Corp.; Carlsbad, Calif.) and Evident Technologies (Troy,N.Y.). The intracellular (pipette) solution (pH 7.3) was composed of1.40 mM Csa, 10 mM EGTA, 10 mM HEPES. The extracellular solution (pH7.4) was composed of 140 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 10mM EGTA, 10 mM glucose, 10 mM HEPES.

In experiments with extracellular delivery of nanoparticles, severaltypes of commercially available nanocrystals were used. in one series ofexperiments, streptavidin-functionalized QD605 (Quantum Dot Corp.) inthe buffer solution B from the QDot® kit were added to extracellularsolution in concentrations from 25 to 500 μg/ml. In another experimentalseries, non-functionalized Maple Red-Orange EviTag-T2 (EvidentTechnologies, Troy, N.Y.) were used in the same concentrations.

For experiments with intracellular applications of nanoparticles,streptavidin-functionalized QD605 (Quantum Dot Corp.) were added to thepipette solution in concentrations from 25 to 500 μg/ml.

Glass 18 mm round coverslips with cells plated on the surface weretransferred into a special chamber 508SW (ALA Scientific Instruments,Westbury, N.Y.). Control extracellular solution was substituted withsemiconductor nanocrystal-containing extracellular solution. After 30minutes at room temperature, the coverslips with cells were washed withPBS solution until excess free-fioating nanocrystals were removed. Tovisually confirm that washing had removed all free-floatingnanoparticles, coverslips were placed under the microscope. Ifexcitation was seen by the naked eye, the washing procedure was repeatedtwo more times.

After the washing procedure was completed, the coverslip was mounted ina microscope chamber and the cells were maintained in buffered EBSSsolution during the experiment. Only cells labeled with nanoparticieswere chosen for further experiments.

Exam le 2 Use of Patch Pipette

Glass micropipettes for patch-clamp experiments were pulled fromborosilicate glass capillaries (1.2 mm no-capillary glass, SutterInstruments; Novato, Calif.) using a Sutter 2000™ pipette puller (modelSutter 2000; Sutter Instruments; Novato, Calif.) using the prerecorded4-step patch pipette pulling protocol. The open diameter of the pipettetip was 1.5-2.2 μm with a resistance of 2-3 MΩ. The micropipettes werefilled with intracellular solution.

Experiments were performed at room temperature in whole-cell patch-clampconfiguration using a Axopatch200B patch-clamp amplifier (MolecularDevices; Sunnyvale, Calif.). After successful giga-seal formation, briefpulses of suction were used to rupture the cellular membrane to achievewhole-cell patch-clamp configuration.

The following test protocol was used for cell stimulation. The membranepotential was set at −70 mV. A depolarizing pulse necessary to take thecell to +40 mV was applied to the interior of the cell for 2 seconds,followed by returning the membrane potential to −70 mV.

Example 3 External Loading of Streptavidin-Coated Nanoparticles

The emission intensity of externally applied streptavidin-functionalizednanoparticles occurring in response to voltage stimulation of the cell(QD605-streptavidin, Quantum Dot Corp.) was visualized using a cooledCCD Optronics Tee 470 camera (Optronic Engineering, Goleta, Calif.)linked to a computer. Voltage changes elicited across the cellularmembrane via patch pipette attached to a cell did not result in changesin the emission intensity of these particular nanoparticles. Nine cellswere tested in this series, and none exhibited changes in emissionintensity to the voltage stimulation protocol described in the previousexample. The streptavidin coating of the nanoparticles used in thisexample may have prevented the nanocrystals from being strategicallyplaced inside the cellar membrane, the site of the highest membranegradient. The streptavidin coating of the nanoparticles used in thisexample may have prevented the nanocrystals from associating with thecellular membrane in such a way that they could effectively monitor thevoltage gradient across the membrane.

Example 4 Intracellular Loading of Nanoparticles

This example was designed to test the emission of nanoparticles loadedintracellularly in response to a voltage change across the cellularmembrane.

It is preferred to position the nanoparticles in close proximity to thecellular membrane in order to achieve modulation of optical signal byvoltage. Since the main part of the voltage gradient exists across thecytoplasmic membrane, the nanoparticles located close to the membrane.would be exposed to a significant portion of the total electricalgradient.

Example 5 Protocol for Intracellular Loading of Nanoparticles

Nanoparticles were added to the patch pipette solution at aconcentration of 200 Initial experiments were performed usingstreptavidin-coated nanoparticles QD605 (Quantum Dot Corp.). A431 cells,plated on glass 18 mm round coverslips were placed into theelectrophysiology chamber mounted on a Zeiss Axiovert 100 microscope.

After establishing a whole-cell patch-clamp configuration, several briefpulses of positive pressure were applied to the pipette interior. Thesesmall changes of intra-pipette pressure were used to facilitate cellperfusion with the intracellular semiconductor nanocrystal-containingsolution, Voltage stimulation experiments on the cells were conductedafter loading of nanocrystals was achieved.

The following test protocol was used for cell stimulation. The membranepotential was set at −70 mV. A depolarizing pulse of +40 mV was appliedto the interior of the cell for 1 to 2 seconds, and subsequently themembrane potential was returned to −70 mV.

Emission of the nanoparticles was recorded constantly during voltagestimulation of the cell using a CCD Optronic Tec 470 camera (OptronicEngineering; Goleta, Calif.). The effects of the voltage stimulation onemission intensity of nanoparticies are shown in FIG. 1. Ten of twelvecells responded to the voltage stimulation protocol, as evidenced by achange in semiconductor nanocrystal emission intensity. Thus, thenanoparticles were able to respond to the changes in transmembranepotential by changing their optical characteristics.

Example 6 Use of Treated Semiconductor Nanoparticles as Voltage Sensors

One prospective use for semiconductor nanoparticle-based membranepotential-sensitive assays is high throughput screening for drugdiscovery, One of the major challenges for HTS assays is the ease ofvoltage indicator loading into cells. Phospholipid-coated quantum dotswere selected as an example of a surface-modified nanoparticle for theseexperiments.

In this example, modified nanoparticles (phospholipid-coated EviTag-T2(Evident Technologies, Troy, N.Y.)) were applied to A431 cellsexternally. Cells attached to 18 mm round coverslips were incubated for45-60 minutes in an extracellular solution containing nanoparticles at25 to 500 μg/ml.

After incubation, the coverslips and attached cells were placed into aspecial chamber 508SW (ALA Scientific Instruments, Westbury, N.Y.) on anZeiss Axiovert 100 microscope, equipped with a CCD camera for opticalrecordings. After establishing a whole-cell patch-clamp configuration asdescribed previously, voltage stimulation experiments were performed.

The following test protocol was used for cell stimulation. Membranepotential was set at −70 mV. A depolarizing pulse of +40 mV was appliedto the interior of the cell for 1 to 2 seconds, and then the membranepotential was returned to −70 mV.

Emission of the nanoparticles was recorded during voltage stimulation ofthe cell using a CCD Optronic Tee 470 camera (Optronic Engineering,Goleta, Calif.). The effects of the electrophysiological stimulation areshown in FIG. 2. Of the 8 cells tested under these experimentalconditions, 6 cells responded to the voltage stimulation protocol bytransiently changing their emission intensity.

These results suggest that nanoparticles having a hydrophobicphospholipid coating can localize in or on the cellular membrane, andtherefore, are able to report on the cellular voltage potential. Thismethod of loading the voltage-sensing nanoparticles represents anespecially advantageous means to prepare cells for high throughputscreening.

Example 7 Summary of Results from Examples 1-6

These results demonstrate that nanoparticles can be used as aself-contained fluorescent voltage indicator. The nanoparticles can beused as a direct optical detection system for changes of the voltagegradient across a membrane. Optimization of delivery and surfacemodifications can further improve the usefulness of the nanoparticles inthe above described methods.

Example 8 Patch-Clamp Recordings from Optically Excited Cells

Cells having an expressed ion channel target can be prepared usingestablished cell culture preparation procedures. CHO or A431 cells,plated on round glass 18 mm coverslips will be incubated with a solutioncontaining nanoparticles at appropriate concentrations for 15-60 minutesat room temperature. After the incubation, the coverslips will be washedfour times with PBS solution.

Alternatively, glass coverslips or plate wells can be pre-coated withthe nanoparticles allowing cells to be seeded on top of the nanoparticlelayer. Wells of the plate will be filled with nanoparticle-containingsolution at the appropriate concentration. The plate can be stored forseveral hours under the sterile conditions.

After the nanoparticles-containing solution is washed away, thecoverslip will be transferred into a special microscope chamber 508SW(ALA Scientific Instruments, Westbury, N.Y.) and maintained in bufferedEBSS solution during the experiment,

Glass micropipettes for patch-clamp experiments will be pulled fromhorosilicate glass capillaries (Sutter 1.2 mm no-capillary glass) usinga Sutter 2000™ pipette puller (model Sutter 2000, Sutter instruments,Novato, Calif.) using a prerecorded 4-step patch pipette pullingprotocol. The open diameter of the pipette tip will be 1.5-2,2 μm.

The micropipettes will be filled with a solution containing 140 mMpotassium aspartate, 5 ruM NaCl, and 10 mM HEPES (pH 7.35). Voltages andcurrents will be recorded at room temperature using a Axopatch 200Bpatch-clamp amplifier (Molecular Devices; Sunnyvale, Calif.).

After establishing the successful Giga-seal, brief pulses of suctionwill be used to rupture the cellular membrane to achieve whole-cellpatch-clamp configuration. The following test protocol will be used forcell stimulation. Brief pulses of excitation light (emitted by laser, orby other light source) will he used to illuminate the patched cell.Voltage and current changes through the cellular membrane will berecorded in the whole-cell configuration.

Example 9 Optical Recordings from Optically Excited Cells

Cells having an expressed ion channel target can be prepared usingestablished cell culture preparation procedures. CHO or A431 cells,plated on round glass 18 mm coverslips will be incubated with a voltagesensitive dye (e.g., a semiconductor nanoparticles-based voltage sensor)for 15-60 minutes. After the incubation, the coverslips will be washedfour times with PBS solution.

The second step will be an incubation of tested cells with a solutioncontaining nanoparticles at an appropriate concentration for 15-60minutes at room temperature. After the incubation, the coverslips willbe washed four times with PBS solution. After the nanoparticle solutionis washed away, the coverslip will be mounted on a microscope chamberand maintained in buffered EBSS solution during the experiment,

Alternatively, glass coverslips or plate wells can be pre-coated withthe nanoparticles allowing cells to be seeded on top of the nanoparticlelayer. Wells of the plate will be filled with nanoparticle-containingsolution at the appropriate concentration. The plate can he stored forseveral hours under the sterile conditions.

Alternatively, at the beginning of experiment the cell suspension willhe incubated with specially prepared suspension of semiconductornanoparticles. After incubating for 5-60 minutes, the cells will hedispensed into wells of a microtiter plate (e.g., a 96, 384, or 836 wellplates). The microtiter plates will be mounted on the microscope stagefor the experiment.

Voltage stimulation will be achieved by illuminating the cell suspensionwith brief pulses of excitation light (emitted by laser, or by otherlight source). Emission of the nanoparticles will be recorded duringvoltage stimulation of the cell using a cooled CCD camera (e.g.,Optronics Tec 470 (Optronic Engineering; Goleta, Calif.) or XR/MEGA-10ZTM fast camera (Stanford Photonics, Inc,; Palo Alto, Calif.)) linked toa computer.

The emission pattern change of the nanoparticies will indicate thecellular response to excitation by photo-activated nanoparticles on thecell surface.

Example 10 First Preparation Method for Target Cells in Microplate Wells

A solution containing non-functionalized Maple Red EviTag-T2 (EvidentTechnologies, Troy, N.Y.) or streptavidin-functionalized QD605nanocrystals (Quantum Dot Corp.) at various concentrations were added tothe 96-well Microplates (Nunc; Denmark). The pretreated plates werestored under sterile conditions for six hours, allowing the solution todry, and leaving the layer of nanoparticles on the bottom of the wells.

Experiments were performed on CHO cells stably expressing M1 muscarinicG_(q)-protein coupled receptor. A suspension of cells was added to theplates and incubated for 12-24 hours at 37° C. in the presence of carbondioxide.

After the incubation, the plates with cells were washed with PBSsolution until any excess free-floating cells and nanocrystals had beenremoved. To confirm that washing had removed all free-floatingnanoparticles, plates were visually inspected with a microscope. ifexcitation was seen by the naked eye, the washing procedure was repeatedtwo more times. After washing, the pates were transferred into PathWay™screening station (BD Biosciences; San Jose, Calif.) for evaluation.

Example 11 Second Preparation Method for Target Cells in MicroplateWells

Cells were plated in 96-well plates. Plates were either glass-bottomedor poly-L-lysine-coated (Nunc; Denmark). Maple Red EviTag-T2nanoparticles were added to the cell-containing solution. Cells wereincubated in the presence of nanoparticles for 15-60 minutes. Any excessnanoparticles were washed away. Plates with nanoparticle-treated cellswere placed inside an environmentally controlled chamber of Pathway HTmachine (BD Biosciences; San Jose, Calif.),

The series of images of cells from each well were acquired in kineticmode from several wells consecutively. First ten images in the serieswere taken as control images to ensure the stability of a signal fromlabeled cells. The following step was an application of potassiumchloride solution into a well. Concentration of potassium chloridesolution was chosen to achieve the final potassium chlorideconcentration of 100 mM thus shifting he membrane potential of cells (toabout 0 mV) in depolarizing direction. The optical response ofnanocrystal-labeled cells to depolarization stimuli for each individualwell was recorded using Pathway HT machine (BD Biosciences; San Jose,Calif.),

After the assays, a series of images were processed using MethaMorphsoftware (Molecular Devices, Sunnyvale, Calif.). Regions of interestwere chosen either around the cellular membrane or in extracellularspace (control).

Example 12 Sensitivity of Externally Applied Nanoparticles to Changes inCellular Membrane Potential Detected by High Content Screening

CHO cells stably expressing MI muscarinic G_(q)-protein coupledreceptors were plated in 96-well microplates, either glass-bottomed orpoly-L-lysine-coated (Nunc; Denmark). Maple Red EviTag-T2 nanoparticleswere added to the cell-containing solution. Cells were incubated in thepresence of nanoparticles for 15-60 minutes. Any excess nanoparticleswere washed away. Plates with nanoparticle-treated cells were placedinside an environmentally controlled chamber of a Pathway HT™ screeningstation (BD Biosciences; San Jose, Calif.).

A series of images of cells from each well were acquired in a kineticmode from several wells consecutively. First, several images in theseries were taken as control images to ensure the stability of a signalfrom labeled cells. Next, potassium chloride solution was added into thewell, The concentration of potassium chloride solution was selected toachieve the final potassium chloride concentration of 100 mM, thusshifting the membrane potential of cells in a depolarizing direction.The optical response of nanocrystal-labeled cells to depolarizationstimuli for each individual well was recorded using a Pathway HT™screening station (BD Biosciences; San Jose, Calif.).

After the. experiments, the series of images was processed usingMethaMorph software (Molecular Devices, Sunnyvale, Calif.). Regions ofinterest (ROI) were selected either around the cellular membrane or inthe extracellular space (control).

Depolarization of cells by extracellular application of potassiumchloride resulted in transient decrease in optical sinal from cells. Itshould be noted that optical signal from extracellular space exhibitedsome intensity decrease as well. However, the effect of potassiumchloride application in cells was significantly higher. For example, inone experiment, change in the maximum response in cellular membrane from12 cells was 349±56 AU, whereas signal intensity change forextracellular space was only 191±38 AU (3 ROIs).

On average, background-subtracted signal intensity in cells decreased17.4±5.1% (number of experiments=8). FIG. 3 represents an example oftransient changes in emission intensity from several cells one well inresponse to cells' exposure to potassium chloride in high concentration.

These results demonstrate that changes in the amplitude of opticalsignal emitted by nanoparticles associated with the cellular membranereflects changes in membrane potential, and confirm that nanoparticlescan act as a sensor of cellular membrane potential.

All of the compositions and/or methods and/or processes and/or apparatusdisclosed and claimed herein can be made and executed without undueexperimentation in light of the present disclosure. While thecompositions and methods of this invention have been described in termsof preferred embodiments, it. will be apparent to those of skill in theart that variations may he applied to the compositions and/or methodsand/or apparatus and/or processes and in the steps or in the sequence ofsteps of the methods described herein without departing from the conceptand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. AU such similar substitutes and modificationsapparent to those skilled in the art are deemed to be within the scopeand concept of the invention.

1. A method for assaying changes in transmembrane potential, the methodcomprising: providing at least one target, wherein the target is a cell,cellular fraction, or artificial membrane structure; contacting thetarget with at least one semiconductor nanocrystal to form a treatedtarget; stimulating the treated target. such that the transmembraneotential of the tar et chanes wherein the at least one semiconductornanocr tal emits light in direct response to a voltage fluctuationresulting from the change in transmembrane potential of the target;assaying emission from the at least one semiconductor nanocrystal; andcorrelating the emission with the change in transmembrane potential. 2.The method of claim 1, wherein the contacting step comprises introducingthe at least on semiconductor into the target.
 3. The method of claim 1,wherein the contacting step comprises introducing the at least onesemiconductor nanocrystal into a cellular membrane of the target.
 4. Themethod of claim 1, wherein the contacting step comprises introducing theat least one semiconductor nanocrystal onto or near a cellular membraneof the target.
 5. The method of claim 1, further comprising assayingemission from the at least one semiconductor nanocrystal after thecontacting step but before the stimulating step.
 6. The method of claim1, wherein the stimulating step comprises electrical stimulation,magnetic stimulation, chemical stimulation, biological stimulation,contacting the target with a drug suspected of being able to activateion channels, contacting the target with a drug suspected of being ableto inhibit ion channels, or combinations thereof.
 7. The method of claim6, wherein the electrical stimulation comprises use of a patch clamp, orapplication of an external electric field.
 8. The method of claim 6,wherein the chemical stimulation comprises contacting the target with apotassium salt or a sodium salt.
 9. The method of claim 6, wherein thebiological stimulation comprises contacting the target with a compoundthat can modify ion channel activity.
 10. The method of claim 1, whereinthe stimulating step comprises maintaining the target at a firstmembrane potential voltage, depolarizing or hyperpolarizing the targetto a second membrane potential voltage, and returning the target to thefirst membrane potential voltage. 11.-14. (canceled)
 15. The method ofclaim 1, wherein the cell is a eucaryotic cell, a procaryotic cell,bacterial cell, a Gram-positive bacterial cell, a Gram-negativebacterial cell, a fungal cell, an insect cell, an avian cell, areptilian cell, an oocyte, a fly cell, a zebrafish cell, a nematodecell, a fish cell, an amphibian cell, or a mammalian cell.
 16. Themethod of claim 1, wherein the cell is a eucaryotic cell.
 17. The methodof claim 1, wherein the cellular fraction is a nucleus, a ribosome, amitochondria, an endoplasmic reticulum, a Golgi apparatus, a vacuole, asynaptic vesicle, or a lysosome.
 18. The method of claim 1, wherein theartificial membrane structure is a phospholipid micelle. 19.-20.(canceled)
 21. The method of claim 1, wherein the at least onesemiconductor nanocrystal is a core-shell nanocrystal.
 22. A method forassaying changes in transmembrane potential, the method comprising:providing at least one cell; contacting the cell with at least onesemiconductor nanocrystal to form a treated cell; stimulating thetreated cell with light; assaying emission from the nanocrystal; andcorrelating the emission with the change in transmembrane potential.23.-29. (canceled)
 30. The method of claim 1, wherein the at least onesemiconductor nanocrystal further comprises a water-compatible surfacecoating.
 31. The method of claim 1, wherein the stimulating stepcomprises stimulating the treated target with light.
 32. The method ofclaim 1, further comprising monitoring transient changes in cellmembrane potential.
 33. The method of claim 1, further comprisingdetecting an optical signal directly from the as least one semiconductornanocrystal.